Opportunistic fungal infections are a major cause of morbidity and mortality in immunocompromised individuals. Candida species are major nosocomial pathogens, and bloodstream infections with these organisms are associated with a mortality rate of 30-40%. Candida albicans and Candida glabrata rank first and second in isolation frequency, respectively, and are responsible for 70% of all cases of disseminated candidiasis. Many strains have acquired resistance to existing drugs, which also have limitations of toxicity, cost, and narrow activity spectra. Thus, there is an urgent need for novel anti-fungal strategies. In this R21 project, which features an interdisciplinary consortium combining research expertise in medicinal chemistry, high-throughput screening (HTS) technology, structural biology, fungal genetics and Candida infection, we propose a new epigenetic approach to anti-fungal agents based on selective inhibition of Candida Bdf1 bromodomains (BDs). Bromodomain and Extra-Terminal (BET) proteins are chromatin-associated factors that regulate gene transcription and chromatin remodeling. BET proteins recognize chromatin through their two BDs (BD1 and BD2), which are small helical domains that specifically bind acetylated lysines on histone peptides. Bromodomain factor 1 (Bdf1) is a fungal BET protein that regulates the transcription of over 500 genes and is essential for correct fungal development and survival. Evidence from high-resolution crystal structures of S. cerevisiae and C. albicans Bdf1-BD1 and BD2 demonstrates that the ligand binding pockets differ significantly from their human BD counterparts, providing the basis for selective inhibition of the fungal BDs. A proof of principle HTS study has identified a family of small molecules sharing a common structural motif, that inhibit C. albicans Bdf1-BD1 (IC50 < 6 M), but do not bind to the corresponding domain from the human ortholog Brd4. In the proposed research, a more extensive library (~1,000,000 compounds) will be screened for strongly bound ligands that are highly selective for both C. albicans and C. glabrata Bdf1-BDs relative to human BDs, using a homogeneous time-resolved fluorescence (HTRF) assay. Hits will be confirmed and IC50 values determined in secondary fluorescence polarization, AlphaScreen, and pull-down assays. Co- crystallization of the most potent inhibitors with the fungal Bdf1-BDs will establish their binding modes, permitting optimization by computer modeling and modified ligand SAR studies. Cell-based assays will establish human cytotoxicity and efficacy in inhibiting fungal growth. This research will provide important new information about fungal Bdf1-BDs in terms of their potential for translation as a novel strategy for drug design within the relatively unexplored field of funga epigenetics, and will create a suite of novel and potent inhibitors for further evaluation in an animal model.